Differential amplification of mutant nucleic acids by pcr in a mixture of nucleic acids

ABSTRACT

A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid; and (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR); wherein the mutant nucleic acid is a G→A mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence to form an AT-rich nucleic acid variant of the parental nucleic acid; and wherein the AT-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T p ) that allows amplification of the parental nucleic acid to thereby enrich the mutant nucleic acid in the nucleic acid mixture.

FIELD OF THE INVENTION

The present invention relates to the differential amplification of mutant nucleic acids by polymerase chain reaction (PCR) in a mixture of nucleic acids. More particularly, this invention relates to preferentially amplifying copies of one or more mutant nucleic acids in a mixture of nucleic adds containing the parental nucleic acid of the mutant nucleic acids in order to enrich the mixture in copies of the mutant nucleic acids.

BACKGROUND OF THE INVENTION

The identification and characterization of mutations have been historically important in every branch of biology and medicine. The study of mutations has contributed significantly to the understanding of the mechanisms and pathways of both normal physiological processes as well as disease pathogenesis. It has now moved on to the study of the relationships between protein structure and function, and correlation between genotype and disease phenotype.

Various developments in molecular genetics and biology have revolutionized the ability to analyze genes at a nucleotide sequence level. The emerging constraint on advances in molecular pathology appears to be the ability to correlate mutant genotype with disease phenotype.

Accordingly, there exists a need in the art to detect small mutations or polymorphisms, involving alterations to one or several bases in a nucleic acid sequence. More particularly, there exists a need in the art to selectively and preferentially produce copies of mutant nucleic acids in mixtures containing the parental nucleic acid so that the mutant nucleic acids can be further analyzed. To these ends, methods should enable the rapid analysis of specific sequences, with decreasing requirements on sample quality and quantity, time, and manual effort.

SUMMARY OF THE INVENTION

Accordingly, this invention aids in fulfilling this need in the art. One embodiment of this invention provides a method for enriching a mutant nucleic acid in a mixture of nucleic acids. The method comprises (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid; and (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR). The mutant nucleic acid is an AT-rich nucleic acid variant of the parental nucleic acid. The AT-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T_(p)) that allows amplification of the parental nucleic acid. The mutant nucleic acid is thereby enriched in the nucleic acid mixture. In one embodiment, the G→A mutant of the parental nucleic acid pairs with a fully complementary nucleic acid sequence to form the AP-rich nucleic acid variant.

This invention also provides a method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid; and (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR). The mutant nucleic acid in this embodiment is a GC-rich nucleic acid variant of the parental nucleic acid. The GC-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T_(p)) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture. PCR is carried out in a reaction medium containing deoxyinosine triphosphate (dITP), or in a reaction medium containing 2,6-diaminopurine triphosphate (dDTP), or in a reaction medium containing dITP and dDTP.

The methods of the invention can include an optional step of detecting the products of the PCR. In addition, the PCR can be carried out in the absence of the parental nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described in greater detail with reference to the drawings in which:

FIG. 1 depicts differential DNA denaturation amplification of G→A-hypermutated HIV-1 genomes. (a) Four sequences harbouring 3, 8, 14 and 18 G→A transitions compared with the reference sequence (0) were amplified under standard PCR conditions with a denaturation temperature of 95° C. M and C denote molecular mass markers and negative control, respectively. 293T/PBMC refers to material amplified from PBMCs infected by an HIV-1Δvif virus stock produced by transfection of 293T cells. (b) The same samples as in (a) were amplified with a denaturation temperature of 83° C. (c) The same PCR products as in (b) were electrophoresed in agarose gel with 1 U HA-yellow (ml agarose)⁻¹. The material for the wild-type control (0) that was not amplified at 83° C. came from lane 0, FIG. 1( a). The black line was added to help to visualize the retardation of AT-rich DNA due to HA-yellow. (d) The relationship between denaturation temperature (T_(d)) and selective amplification was explored by using another series of clones with 0, 5, 10, 15, 20 and 24 G→A transitions with respect to the reference sequence (0).

FIG. 2 depicts a collection of G→A-hypermutated HIV-1 V1V2 region sequences derived from Δvif stock virus grown on 293T cells [293T/PBMC, FIG. 1( a-c)]. For clarity, only a 189 bp region of the 304 bp segment that was amplified is shown. Sequences are aligned with respect to the parental sequence. Only differences are shown. Hyphens denote gaps. Clone designation is shown to the left. Analysis of material from the 95° C. amplification failed to identify any hypermutated genomes.

FIG. 3 depicts a collection of AT-rich poliovirus VP1 segments derived from a patient with post-vaccinal acute flaccid paralysis. For clarity, only a 109 bp region of the 480 bp segment that was amplified is shown. Sequences are aligned with respect to poliovirus Sabin 1. Only differences are shown. Clone designation is shown to the right. The 3D-PCR-amplified segments bore one to six GC→AT transitions compared with Sabin 1. Analysis of material from the 95° C. amplification yielded two substitutions among 17 clones in the same sequence.

FIG. 4A depicts the sequences of two alleles of the p21 ras gene. Primers are underlined in the Figure.

FIG. 4B depicts primers for amplifying a 19 bp window of the p21 ras gene.

FIG. 5 depicts the chemical structure of dUTP and dCTP analogues that can optionally be substituted for dTTP.

DETAILED DESCRIPTION OF THE INVENTION

It is one of the truisms of genetics that adenosine (A) pairs with thymine/uracil (T/U) while guanosine (G) pairs with cytidine (C). Pairing involves non-covalent hydrogen bonds, two for the A:T pair, three for the G:C pair.

Virus genomes from the same family may exhibit a wide range in their DNA GC content, whereas viral hypermutants differ substantially in GC content from their parental genomes. As AT-rich DNA melts at lower temperatures than GC-rich DNA, use of a lower denaturation temperature during PCR should allow differential amplification of AT-rich genomes or variants within a quasispecies. The latter situation has been explored explicitly in a two-step process by using a series of well-defined viral sequences differing in their AT content. Firstly, the lowest denaturation temperature (T_(p)) that allowed amplification of the parental sequence was determined. Secondly, differential amplification of AT-rich viral variants was obtained by using a denaturation temperature 1-3° C. lower than T_(p). Application of this sensitive method to two different viruses made it possible to identify human immunodeficiency virus type 1 G→A hypermutants in a situation where none were expected and to amplify AT-rich variants selectively within a spectrum of poliovirus mutants.

Thus, method according to this invention allows differential amplification of DNA segments differing by one to many GC->AT transitions. As the degree of substitution directly impacts the melting temperature of the DNA, the lower the denaturation temperature the more substituted the genomes amplified. As different loci may have widely different base compositions the conditions can be optimized for each segment. The method of the invention applies to DNA or cDNA (reverse transcribed RNA) no matter the origin. Preferred sources of nucleic acids are HIV-1, HIV-2, poliovirus, and measle virus.

In its broadest sense, this invention relates to the amplification of segments of DNA by the polymerase chain reaction (PCR). As used herein, the terms “polymerase chain reaction” and “PCR” are used in their conventional sense as an in vitro method for the enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in a target DNA. A repetitive series of cycles involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5′ ends of the primers. Because the primer extension products synthesized in one cycle can serve as templates in the next cycle, the number of target DNA copies approximately doubles at every cycle. The use of the thermostable DNA polymerase, such as Taq polymerase isolated from Thermus aquaticus, makes it possible to carry out the PCR reaction of the invention in a simple and robust manner, which can be automated using a conventional thermal cycling device.

The PCR reaction of the invention can be carried out using conventional reaction components, such as, the template DNA, primers, Taq or another polymerase, dNTP's, and buffer. The reaction can be carried out in the conventional manner by simply cycling the temperature within a reaction chamber. The specificity and yield of the amplification reaction can be regulated by controlling well-known parameters, such as enzyme, primer, dNTP, and Mg⁺⁺ concentrations, as well as the temperature cycling profile.

Because of the wide variety of applications in which PCR is used according to the invention, it is not possible to describe a single set of conditions for all situations. The amplification can be initially performed in a DNA Thermal Cycler (Perkin-Elmer Cetus Instruments) using the “Step-Cycle” program and reagents recommended by the manufacturer. For any given pair of oligonucleotide primers an optimal set of conditions can then be established.

Once the reagents and the step-cycle program have been established for the target nucleic acid sequence being amplified, the lowest denaturation temperature that allows amplification of the parental sequence is determined. This lowest denaturation temperature is termed T_(p). Differential amplification of AT-rich nucleic acid variants is obtained by using the same reagents and the same step-cycle program, except that the denaturation temperature for each cycle of PCR is about 1 to 3° C., preferably 1° C., lower than T_(p). Thus, the temperature employed for the denaturation step of PCR will be a temperature at which the mutant nucleic acid is preferentially amplified relative to the parental nucleic acid. Preferential amplification can be determined, for example, by gel electrophoresis or by direct sequencing of PCR products or by detection with a labeled probe.

The nucleic acid mixture employed in the methods of the invention can comprise a parental nucleic acid and at least one mutant nucleic acid of the parental nucleic acid. The mutant nucleic acid can contain at least one G→A mutation relative to the parental nucleic acid to form, after base pairing, an AT-rich nucleic acid variant of the parental nucleic acid. Alternatively, the mutant nucleic acid can contain at least one A→G mutation relative to the parental nucleic acid to form, after base pairing, a GC-rich nucleic acid variant of the parental nucleic acid. It will be understood that the mixture can contain a mutant nucleic acid having both G→A and A→G mutations at different loci, or the mixture can contain two or more nucleic acid mutants each containing either one or more G→A mutations or one or more A→G mutations. The number of mutations in the mutant nucleic acids is typically 1-18 mutations compared to the parental nucleic acid.

The methods of the invention apply to any DNA or cDNA (reverse transcribed RNA) fragment no matter the origin. An important property of the PCR reaction of the invention, particularly in diagnostic applications, is the capacity to amplify a target sequence from crude DNA preparations as well as from degraded DNA templates. The DNA in the sample to be amplified need not be chemically pure to serve as a template provided that the sample does not contain inhibitors of the polymerase. The ability to amplify specific sequences from crude DNA samples has important implications for research applications, for medical diagnostic applications, and for forensics.

The primers used in PCR can contain mismatches relative to the sequences to which they base-pair. For example, the primers can be degenerate, as is described for primers SK122/SK123 used to hybridize with the V1V2 region of the HIV-1 envelope gene in the Examples hereinafter. If the primers contain mismatches relative to the sequence to which they base-pair, the hybridization step of PCR can be optimized independently of the denaturation step of PCR. In addition, it will be understood that the primers can contain mismatches relative to the parental sequence.

The length of the primers has not been found to be critical in carrying out the methods of the invention. Standard length primers can be employed, and optimal primer length can be determined by routine experimentation. Typically, the primers will be about 20-25 bp, but may be longer or shorter.

Similarly, the length of the parental sequence has not been found to be critical in carrying out the methods of the invention. Typically, the parental sequence will be up to about 500 bp. It will be understood that longer or shorter sequences can be employed.

Further, the size of the mutant nucleic acids being amplified has not been found to be critical in the methods of the invention. Mutant nucleic acids up to about 500 bp can be employed, although it will frequently be more convenient to use shorter sequences.

The region of the mutant nucleic acids being, amplified, also referred to as the window between the two primers, can vary depending upon the target nucleic acids. For example, the region amplified can comprise about 20, 30, 40, 50, or 60 bp, although longer or shorter sequences are contemplated by the invention. Amplified regions of 19 and 30 bp are described for p12 ras gene in the Examples hereinafter.

In some cases, the amplified region may affect the manner in which the amplified nucleic acids are detected. For example, to detect a single point mutation, the window between two primers can be 3-12 nucleotides, but in this case, using 20 bp primers, the bands of the PCR products are about 43-52 base pairs. Nucleic acid molecules of this size can not readily be detected by electrophoresis in agarose gel, but they can be detected in polyacrylamide gel.

In any event, the detection method can be adapted to the characteristics of the amplified PCR product. Preferred detection methods are gel electrophoresis in agarose or acrylamide gel, capillary electrophoresis, or chromatography, especially gel filtration or ion-exchange chromatography.

The methods of this invention have a wide variety of uses. For example, the methods can be employed to characterize the origin of parental DNA or to detect mutations characteristic of human gene disorders. The methods can also be employed for detecting G→A mutant strains of HIV, particularly G→A hypermutants, that are resistant to anti-retroviral drugs. Further, the methods of the invention can be employed for detecting neurovirulent vaccine-derived poliovirus isolates that cause vaccine-associated paralytic poliomyelitis. For bibliographic references about the nucleotide variation of vaccine strains of poliovirus see Buttinelli et al, J. Gen. Virol. 2003, 84, 1215-1221 and Georgescu et al., J. Virol. 1997, 71, 7758-7768. In a preferred embodiment of the invention, the methods of the invention are used to amplify parental and mutant nucleic acids from measle virus.

It will be understood that the methods of the invention are useful for detecting specific mutations at specific sites, which have previously been characterized and sequenced. The methods of the invention are also useful for detecting the presence of unknown sequence differences in a given length of DNA. The methods are useful for detecting known mutations, and are particularly useful for rapidly detecting multi-allelic loci. It will be understood that the methods of the invention can also be used to amplify sequences containing small deletions, such as 1 or 2 bp deletions.

Although the method allows differential amplification, it is not quantitative per se. However, coupled to limiting dilution of input DNA it is possible to quantitate the fraction of AT rich genomes within a sample. Alternatively, Taqman PCR can be performed at 95° C. and the selective temperature to determine the copy number per sample. The ratio of the two values can give the relative concentration of AT-rich alleles with respect to the total concentration of all alleles.

The methods according to the invention encompass variants that use modified bases that can influence slightly the melting temperature of DNA. For example, dUTP can be used to replace dTTP (FIG. 5). The difference will be small. In the same vein, one can use modified derivatives of dCTP, such as 5-methyl dCTP, 5-fluoro dCTP, 5-chloro dCTP, 5-bromo dCTP, or 5-iodo dCTP, which can be incorporated into DNA by Taq polymerase or another thermostable DNA polymerase (FIG. 5). These dCTP analogues are particularly interesting, for when incorporated into DNA, the DNA melts to higher temperatures (Hoheisel et al., 1990, Wong et al., 1991). Accordingly, use of any one of these modified bases will enhance the discrimination between the parental sequence and AT rich allele.

Equally, the methods of the invention encompass the use of non-standard PCR buffer conditions, particularly the use of certain salts and salt concentrations and the use of organic molecules. It is well known that the denaturation temperature can be influenced by the nature of the ion and ionic strength, for example tetraethylammonium chloride (Muraoka et al., 1980) and the use of small organic molecules, such as methanol or polyethylene glycol, to cite just two (Muraoka et al., 1980, Votavova et al., 1986).

The methods of the invention can be used to detect small deletions in an allele. The melting temperature is a function of the number of hydrogen bonds distinguishing two alleles. Deletion of a single base will remove 2 or 3 hydrogen bonds, and hence will melt to lower temperature. Larger deletions will be detected more readily. Hence, the method of the invention can be used to selectively amplify alleles with small deletions, for example, mitochondrial DNA or microsatelites associated with a disease susceptibility gene, although these are mentioned as examples and not to limit the invention.

By reducing the size of the PCR fragment to 60-80 bases, alleles differing by a single GC->AT substitution can be differentially amplified. Hence, the method of the invention can be used in the search for single nucleotide polymorphisms (SNPs). Being temperature based, no allele specific oligonucleotides are necessary.

As the window of observation is generally between 10-30 bases (60-80 bp-2×25 bp primers), the method of the invention can detect any mutation provided that it reduces the melting temperature of the allele. As GC->AT substitutions represent the most frequent substitutions (40-50%) characterizing human gene disorders, p53 inactivating mutations or those of pseudogenes compared to the orthologous gene, it is possible to apply the method of the invention to the detection of a mutation characteristic of a pre-tumourous cell in a blood sample or in the characterization of human genotypes via SNP typing.

The methods of the invention, designated “3DPCR,” can be used to selectively amplify AT rich alleles from the normal counterpart. This follows on from the fact that an A:T base pair involves 2 hydrogen bonds, while a G:C pair involves 3 (FIG. 1). Consequently, it has been demonstrated that the denaturation temperature of an AT rich allele will be slightly lower than that of the normal counterpart. The converse, the selective amplification of a G:C rich allele compared to a normal counterpart, is not amenable to analysis by 3DPCR because it would melt to a higher temperature than the normal allele, and at higher temperatures both the normal and GC rich alleles will be amplified.

This invention provides another method that addresses this problem. Deoxyinosine triphosphate (dITP) can be substituted for dGTP in PCR. Inosine lacks the amino group at position 2 compared to guanosine. As a consequence, dITP forms only 2 hydrogen bonds with dCTP (FIG. 5). Hence, the specificity of base pairing is preserved (G or I pairs with C), and with it the information, while the number of hydrogen bonds is reduced from 3 to 2. By contrast, 2,6-diamino purine (D) bears an additional amino group compared to adenosine (2-aminopurine). Furthermore, it base pairs with thymidine via 3 hydrogen bonds (FIG. 5). Again the information content is preserved (D or A pairs with T).

Thus, while it is known that dDTP and dITP can substitute for dATP and dGTP in a PCR reaction, indeed both can be used in the same reaction, PCR material so derived will have the inverse melting properties compared to products bearing the canonical bases, dATP and dGTP. G:C rich alleles, (I:C in fact, 2 hydrogen bonds) will melt to slightly lower temperatures than the normal A:T allele (D:T in fact, 3 hydrogen bonds).

Accordingly, performing 3DPCR of the invention with these modified bases allows selective amplification of A:T rich alleles. The invention thus concerns the use of a lower melting temperature to amplify a subset of alleles that can be distinguished by a fractionally lower DNA melting temperature. Accordingly, this invention also involves the use of dDTP and dITP as a means to convert a G:C rich allele into DNA that melts at a lower temperature. This method of the invention is termed “inverse 3DPCR” or “i3DPCR” to emphasize that it allows amplification of G:C rich alleles as opposed to A:T rich alleles, which 3DPCR does.

There is much renewed interest recently in adenosine deamination of viral RNA. This results from editing of adenosine residues in RNA by an interferon induced host cell enzyme, ADAR, and its isoforms. The enzyme deaminates A to yield inosine (I). When repeated, this gives rise to A->G hypermutants for, as mentioned above, I pairs as G.

The i3DPCR method according to the invention is well adapted to selectively amplifying such viral hypermutants. The viral paradigm concerns measles virus hypermutants. However, to much lower degrees, A->G hypermutants have been described for parainfluenza virus, respiratory syncytial virus, vesicular stomatitis virus, and some retroviruses including HIV. Thus, the method of the invention is useful in basic research involving these and other diseases.

The i3DPCR method allows differential amplification of DNA segments differing by one to many AT->GC transitions. As the degree of substitution directly impacts the melting temperature of the DNA, the lower the denaturation temperature the more substituted the genomes amplified. As different loci may have widely different base compositions the conditions can be optimized for each segment.

Although the i3DPCR method allows differential amplification, it is not quantitative per se. However, coupled to limiting dilution of input DNA, it is possible to quantitate the fraction of GC rich genomes within a sample. Alternatively, Taqman PCR can be performed at 95° C. and the selective temperature to determine the copy number per sample. The ratio of the two values can give the relative concentration of GC-rich alleles with respect to the total concentration of all alleles.

The i3DPCR method encompasses variants that use modified bases that can influence slightly the melting temperature of DNA. For example 5-bromodUTP or dUTP can be used to replace dTTP. The differences may be small. Accordingly, use of any one of these modified bases will enhance the discrimination between the parental sequence and GC-rich allele.

Equally, the i3DPCR methods cover the use of non-standard PCR buffer conditions, particularly the use of certain salts and salt concentrations and the use of organic molecules. It is well known that the denaturation temperature can be influenced by the nature of the ion and ionic strength, for example, tetraethylammonium chloride (Muraoka et al., 1980), and the use of small organic molecules, such as methanol or polyethylene glycol, to cite just two (Muraoka et al., 1980, Votavova et al., 1986).

If random PCR is performed using a low denaturation temperature, i3DPCR can amplify GC-rich DNA from genomes. G:C rich DNA is usually synonymous with coding regions and can help in identifying genes within genomes.

More importantly, and like 3DPCR, i3DPCR can be used to identify single point mutations in a small window. Already 3DPCR has been used to identify a single G->A base change in a small locus of between 60-80 base pairs (bp). Similarly, i3PCR can identify a single A->G or T->C mutation in a small locus of 60-80 bp. Hence, the i3DPCR method can be used to identify alleles with G/C rich point mutations within a mass of normal A:T alleles. The obvious example is to look for mutations characteristic of a pre-tumoral cell.

Together 3DPCR and i3DPCR can pick up 85% of all mutations characteristic of human gene disorders or p53 inactivating lesions (Krawczak et al., 1995, Li et al., 1984). The 15% of remaining mutations concern G<->C and A<->T transversions. While the number of base pairs is not altered by the mutations, it is possible that stacking energies will be affected, which could lead to a change in melting temperature. If so, then 3DPCR and i3DPCR can be used to identify these mutations too.

This invention will be described in greater detail in the following Examples.

Example 1

The human immunodeficiency virus (HIV) Vif protein intercepts the host-cell proteins APOBEC3F and APOBEC3G, preventing their incorporation into budding virions (Harris et al., 2003; Wiegand et al., 2004; Zheng et al., 2004). The resulting Vif/APOBEC3 complexes are shunted to the proteasome for degradation (Sheehy et al., 2003; Yu et al., 2003). Of the seven APOBEC3 genes on human chromosome 22, at least five are transcribed (Jarmuz et al., 2002; http://genecards.bcgsc.bc.ca). They belong to a group of cytidine deaminases; the prototype of these is APOBEC1, which specifically edits the apolipoprotein B mRNA in the environment of the intestine (Teng et al., 1993). Although mRNA-editing functions have not yet been ascribed to any APOBEC3 molecule, APOBEC3C, -3F and -3G are able to extensively deaminate single-stranded DNA (Harris et al., 2003; Lecossier et al., 2003; Suspène et al., 2004; Wiegand et al., 2004; Yu et al., 2004).

In the singular context of a HIV Δvif virus, only APOBEC3F and -3G appear to be packaged into the virion (Harris et al., 2003; Bishop et al., 2004; Liddament et al., 2004; Wiegand et al., 2004; Zheng et al., 2004). It is of note that APOBEC3F and -3G are packaged during budding from the donor cell and do not enter the replication complex of an incoming virion. Consequently, as soon as minus-strand viral cDNA is synthesized in the next round of infection, the numerous multiple C residues are deaminated, yielding U. Following plus-strand DNA synthesis, the U residues are copied into A, giving rise to so-called G-4A hypermutants, by reference to the viral plus strand (Pathak & Temin, 1990; Vartanian et al., 1991).

As G→A hypermutants are associated with a lethal phenotype, the absence of vif, their detection in a natural setting is, not surprisingly, highly variable and their frequency is often low. However, as GRA hypermutants frequently exhibit 20-60% of G residues substituted by A, their base composition is shifted considerably from that of the parental sequence. Hence, there is a need for a method that allows amplification of AT-rich variants and not the parental sequence.

From previous work on HIV GRA hypermutation, a large collection of molecular clones was available, corresponding to the V1V2 region of the HIV-1 envelope gene, that differed uniquely in the number of G→A transitions. A smaller region within this fragment was amplified by using Taq polymerase and degenerate primers that were derivatives of the standard SK122/SK123 pair (Goodenow et al., 1989), to result in better amplification of hypermutated genomes. Their sequences were: SK122intD, 5′-AAARCCTAAARCCA TRTRTA [SEQ ID NO: 1]; SK123intD, 5′-TAATGTATGGGAATTGGYTYAA [SEQ ID NO: 2]. When the PCR denaturation temperature was lowered to 83° C. (the reaction profile was 5 min at 83° C., 25 cycles of 1 min at 83° C., 30 s at 45° C. and 30 s at 72° C., followed by 10 min at 72° C.), it was possible to uniquely amplify clones harbouring at least three mutations, whilst not amplifying the parental sequence (no mutations; FIGS. 1 a, b).

Example 2

To confirm that amplified material was indeed hypermutated and not a PCR artefact, products were electrophoresed in agarose gel containing HA-yellow (Hanse Analytik), a pegylated bisbenzamide that interacts preferentially with the minor groove of AT-rich DNA, thus retarding migration (Abu-Daya et al., 1995; Abu-Daya & Fox, 1997; Janini et al., 2001). As can be seen in FIG. 1( c), migration of PCR products in a gel containing 1 U HA-yellow (ml agarose)⁻¹ was retarded progressively when moving from 0 to 18 transitions per sequence, confirming the selective amplification of G→A-hypermutated DNA at 83° C.

Example 3

It is apparent from FIG. 1( b) that product recovery correlated with the extent of hypermutation. To explore more carefully the relationship between denaturation temperature and the number of G→A transitions per clone, another series of G→A-hypermutated reference clones spanning another locus within the V1V2 region was analyzed by using Taq polymerase and a different pair of primers, RT3 and RT4 (Martinez et al., 1994). Lowering the denaturing temperature by 1° C. progressively amplified more extensively hypermutated sequences (FIG. 1 d). Given the exquisite relationship between denaturation temperature and AT content of a sequence, the success of amplification may also depend on the calibration of the PCR machine and perhaps upkeep and make. Accordingly, all PCRs were performed on the same machine.

These findings show that the selective amplification of G→A hypermutants is indeed generally related to the melting temperature of the target DNA. This method of the invention is referred to as differential DNA denaturation PCR, or 3D-PCR.

Example 4

FIG. 1 also shows nested PCR material (293T/PBMC) corresponding to the same V1V2 region amplified from peripheral blood mononuclear cells (PBMCs) that had been infected with a Δvif derivative of HIV-1 pNL4.3 following transfection of 293T cells. The denaturation temperature was 83° C. The fact that this material represented differentially amplified G→A hypermutants was indicated when the 3D-PCR products were electrophoresed in a gel containing HA-yellow (FIG. 1 c, 293T/PBMC). When the 3D-PCR products were cloned and sequenced, the vast majority of sequences were extensively hypermutated, harbouring between three and 18 GRA transitions compared with the reference sequence (FIG. 2). Of the 18 sites bearing G→A transitions, 15 were in the context GpA and PCR material amplified at 95° C. identified only wild-type DNA (not shown).

The surprise here is that the HIV-1Δvif virus stock was made by using the 293T cell line, which is widely used as not only can it be transfected easily, but also it is considered not to express APOBEC3 molecules. From what is known of the mechanism of G→A hypermutation, the simplest explanation is that the 293T cell line had become clonally heterogeneous, so that APOBEC3F [preference for 5′TpC dinucleotide, GpA on viral plus strand (Harris et al., 2003; Liddament et al., 2004; Wiegand et al., 2004; Zheng et al., 2004)] as opposed to APOBEC3G [(5′CpC preference, or GpG on plus strand (Harris et al., 2003; Lecossier et al., 2003; Suspène et al., 2004)] was being expressed in a subset of cells. Presumably 3D-PCR was picking up DNA from viruses produced by this subset.

Example 5

Poliovirus VP1 PCR products from ten patients with post-vaccinal acute flaccid paralysis (Balanant et al., 1991) were examined. A smaller 480 bp nested segment was targeted and the denaturation conditions were investigated by using the primer pair UG1/UC1 (Guillot et al., 2000). Calibration using cloned DNA showed that the reference Sabin 1 sequence was amplified by using denaturation temperatures from 95 to 91° C., but not from 90 to 80° C. Sabin 2 and 3 targets were subtly different from Sabin 1 in that they could not be amplified below 92° C. The higher denaturation temperature used here compared with the HIV-1 locus described earlier is explained by the higher GC content of the target (48%, compared with 34% for HIV-1). Among the ten samples, only one yielded a strong signal by 3D-PCR, with the following reaction profile: 5 min at 90° C., 25 cycles of 1 min at 90° C., 30 s at 45° C. and 30 s at 72° C., followed by 10 min at 72° C. When cloned and sequenced, a series of AT-enriched sequences was obtained, with substitutions mapping particularly to VP1 residues 560-728 in the alignment of enteroviral polyproteins (www.iah.bbsrc.ac.uk/virus/picornaviridae/SequenceDatabase/alignments/entero_pep.txt). The sequences carried between one and six substitutions per segment. Of the 34 distinct substitutions, 28 were non-synonymous (including two nonsense), which is typical of variation within a quasispecies that has not undergone purifying selection. All but one substitution yielded genomes that were enriched in A and T. Amplification, cloning and sequencing of PCR material obtained at 95° C. revealed 17 clones that harboured only two substitutions in the locus shown in FIG. 3 (data not shown). Hence, it can be concluded that 3D-PCR was indeed amplifying the AT-rich end of the poliovirus mutant spectrum. As only one sample could be amplified differentially, the AT-rich variants presumably represent an unusually broad mutant spectrum and have nothing to do with the post-vaccination syndrome.

Example 6

The length of the window affects the ability to discriminate between alleles differing in GC content. The longer the DNA segment the poorer the discrimination. The inverse is true to the point that an attempt was made to identify a single point mutation in a small window of as little as 30 bases. The case chosen was the p21 ras gene and the “famous” mutation in codon 12 that transforms the gene into an oncogene. The sequences of the two alleles are shown in FIG. 4A. The PCR primers are underlined as is the single G residue in the wild type sequence that is mutated to T in the oncogene. The “window” between the two primers is 29 bp.

Under standard PCR conditions, it is clear that the wild type allele was amplified at temperatures equal to and greater than 89° C. By contrast, the mutant allele could be amplified at temperatures equal to and greater than 88° C. Hence, it is indeed possible to selectively amplify alleles on the basis of a single GC->AT substitution.

The impact of amplifying across an even smaller window of 19 bp was explored. The primers are shown in FIG. 4B. Again using standard PCR conditions, it was possible to distinguish the mutant and wt alleles. As expected for a smaller DNA fragment (69 bp compared to 79 bp), the denaturation temperatures were lower. At 86.2° C., only the mutant allele could be identified, whereas at 87.3° C., both the mutant and wild type alleles were amplified (not shown). Once again approximately one degree is sufficient to allow differential amplification.

Although technically feasible in terms of PCR, going below 69 bp may prove inconvenient for analysis by agarose gel electrophoresis as it becomes increasingly difficult to distinguish the band from that of primer-dimers. However, acrylamide gel electrophoresis, which is somewhat less convenient, is capable of distinguishing between smaller bands.

In summary, the methods of the invention, namely, 3D-PCR can be used to differentially amplify AT-enriched genomes compared with the parental genome. Although retroviral hypermutants are preferred targets for 3D-PCR, it can be applied to any sample in which there is a mutant or mutant spectrum.

3D-PCR allows differential amplification of genomes that differ by just a few GC→AT transitions. As the degree of substitution directly affects the melting temperature of the DNA, the lower the denaturation temperature, the more substituted the genomes that are amplified. As different loci may have widely different base compositions, the conditions can be optimized for each segment. Although the method allows differential amplification, it is not quantitative per se. However, coupled to limiting dilution of input DNA, it is possible to quantify the fraction of AT-rich genomes within a sample. 3D-PCR can be used to amplify AT-rich bacterial 16S rDNA sequences within a heterogeneous natural sample, neo-deaminated immunoglobulin V regions, or promoter regions that have undergone extensive 5-MeC deamination following extensive methylation.

In the precise setting of HIV, 3D-PCR has shown that one cell line that is used widely to support the replication of Δvif genomes is probably clonally heterogeneous, meaning that there is a background G→A-hypermutated signal in any sample. The ability to discriminate AT-rich variants over background indicates that this technique can be employed in a variety of applications to biological questions.

REFERENCES

The entire disclosures of each of the following publications are relied upon and incorporated by reference herein.

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1. A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid; (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR) using a pair of PCR primers; and (c) optionally detecting the products of the PCR; wherein the distance between the pair of primers in the PCR of step (b) is 3-30 bases; wherein the mutant nucleic acid differs from the parental nucleic acid in 1 or 2 bases; wherein the mutant nucleic acid comprises a greater number of adenine (A) and thymine (T) nucleotides than the parental nucleic acid; and wherein the mutant nucleic acid is selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T_(p)) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture.
 2. The method as claimed in claim 1, wherein the mutant nucleic acid is a G/C→A/T mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence.
 3. The method as claimed in claim 1, wherein the mutant nucleic acid is a small deletion mutant of the parental nucleic acid.
 4. The method as claimed in claim 3, wherein the deletion comprises 1 or 2 bp.
 5. The method as claimed in claim 1, wherein the nucleic acid mixture contains the parental nucleic acid and one G/C→A/T mutant of the parental nucleic acid, which is selectively amplified compared to amplification of the parental nucleic acid.
 6. The method as claimed in claim 1, wherein the nucleic acid mixture contains the parental nucleic acid and more than one G/C→A/T mutant nucleic acid of the parental nucleic acid, wherein each mutant nucleic acid is selectively amplified compared to amplification of the parental nucleic acid.
 7. The method as claimed in claim 1, wherein the nucleic acid mixture contains the parental nucleic acid and one G/C→A/T mutant nucleic acid of the parental nucleic acid, in which up to 60% of the G/C residues have been substituted by A/T in the parental nucleic acid, and wherein mutant nucleic acids in the mixture are selectively amplified compared to amplification of the parental nucleic acid.
 8. The method as claimed in claim 1, wherein products of the PCR are detected by gel electrophoresis in agarose or acrylamide gel, capillary electrophoresis, or chromatography.
 9. The method as claimed in claim 1, wherein the products of the PCR are detected by gel filtration or ion-exchange chromatography.
 10. The method as claimed in claim 6, wherein products of the PCR are identified by relative location in the gel.
 11. The method as claimed in claim 1, wherein the mutant nucleic acid is a hypermutated variant of the parental nucleic acid in the nucleic acid mixture and the denaturing temperature is about 1° C. lower than T_(p).
 12. The method as claimed in claim 2, wherein the mutant nucleic acid contains 1 or 2 G/C→A/T mutations compared to the parental nucleic acid.
 13. The method as claimed in claim 1, wherein step b) of amplification of nucleic acids by PCR is carried out with modified bases.
 14. The method as claimed in claim 13, wherein the modified bases are dUTP, 4-methyl dCTP, 5-bromo dCTP, or 5-iodo dCTP, or mixtures thereof.
 15. The method as claimed in claim 1, wherein step b) of amplification of nucleic acids by PCR is carried out with non-standard PCR buffer comprising tetraethyl-ammonium chloride, methanol, or polyethylene glycol.
 16. The method as claimed in claim 1, wherein the parental nucleic acid comprises 40 to 500 bases.
 17. The method as claimed in claim 1, wherein the parental nucleic acid comprises 40 to 80 bases.
 18. The method as claimed in claim 1, wherein the parental nucleic acid is a viral nucleic acid.
 19. The method as claimed in claim 18, wherein the parental nucleic acid is chosen from HIV, poliovirus, and measle virus nucleic acid.
 20. The method as claimed in claim 19, wherein the parental nucleic acid is a poliovirus nucleic acid.
 21. A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid; (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR) using a pair of PCR primers; and (c) optionally detecting the products of the PCR; wherein the distance between the pair of primers in the PCR of step (b) is 3-30 bases; wherein the mutant nucleic acid differs from the parental nucleic acid in 1 or 2 bases; wherein the mutant nucleic acid comprises a greater number of guanine (G) and cytosine (C) nucleotides than the parental nucleic acid; and wherein the mutant nucleic acid is selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T_(p)) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture; and wherein PCR is carried out in a reaction medium containing deoxyinosine triphosphate (dITP), or in a reaction medium containing deoxy 2,6-diaminopurine triphosphate (dDTP), or in a reaction medium containing dITP and dDTP.
 22. The method as claimed in claim 21, wherein the mutant nucleic acid is an A/T→G/C mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence.
 23. (canceled)
 24. A method for characterizing the origin of parental DNA or for detecting mutations characteristic of human gene disorders, wherein the method comprises enriching a mutant nucleic acid in a mixture of nucleic acids according to the method as claimed in claim 1 and then further analyzing the products to determine the origin of parental DNA or to detect mutations characteristic of human gene disorders.
 25. A method for detecting a G→A mutant strain of HIV (G→A hypermutants) that is resistant to antiretroviral drug, wherein the method comprises enriching a mutant nucleic acid in a mixture of nucleic acids according to the method as claimed in claim 1 and then further analyzing the products to detect a G→A mutant strain of HIV that is resistant to antiretroviral drug.
 26. A method for detecting neurovirulent vaccine-derived poliovirus isolates that cause vaccine-associated paralytic poliomyelitis, wherein the method comprises enriching a mutant nucleic acid in a mixture of nucleic acids according to the method as claimed in claim 1 and then further analyzing the products to detect neurovirulent vaccine-derived poliovirus isolates that cause vaccine-associated paralytic poliomyelitis.
 27. The method as claimed in claim 21, wherein the parental nucleic acid is a viral nucleic acid.
 28. A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: (a) providing a nucleic acid mixture comprising a mutant nucleic acid of a parental nucleic acid; (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR) using a pair of PCR primers; and (c) optionally detecting the products of the PCR; wherein the distance between the pair of primers in the PCR of step (b) is 3-30 bases; wherein the mutant nucleic acid differs from the parental nucleic acid in 1 or 2 bases; wherein the mutant nucleic acid comprises a greater number of adenine (A) and thymine (T) nucleotides than the parental nucleic acid; and wherein the mutant nucleic acid is selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T_(p)) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture.
 29. A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: (a) providing a nucleic acid mixture comprising a mutant nucleic acid of a parental nucleic acid; (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR) using a pair of PCR primers; and (c) optionally detecting the products of the PCR; wherein the distance between the pair of primers in the PCR of step (b) is 3-30 bases; wherein the mutant nucleic acid differs from the parental nucleic acid in 1 or 2 bases; wherein the mutant nucleic acid comprises a greater number of guanine (G) and cytosine (C) nucleotides than the parental nucleic acid; and wherein the mutant nucleic acid is selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T_(p)) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture; and wherein PCR is carried out in a reaction medium containing deoxyinosine triphosphate (dITP), or in a reaction medium containing deoxy 2,6-diaminopurine triphosphate (dDTP), or in a reaction medium containing dITP and dDTP.
 30. The method as claimed in claim 27, wherein the parental nucleic acid are chosen from HIV, poliovirus, and measle virus nucleic acids.
 31. A method for characterizing the origin of parental DNA or for detecting mutations characteristic of human gene disorders, wherein the method comprises enriching a mutant nucleic acid in a mixture of nucleic acids according to the method as claimed in claim 21 and then further analyzing the products to determine the origin of parental DNA or to detect mutations characteristic of human gene disorders. 